Many neurological disorders result from the loss of neurons through disease or injury, and these cells are not intrinsically replaced. Such neurological disorders include neurodegenerative disorders of the CNS, such as Parkinson's disease (PD), Alzheimer's disease (AD), multiple sclerosis, Huntington's disease and amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease). AD and ALS are due to the degeneration of cholinergic neurons. Such neurological disorders also include neurotrauma, such as spinal cord injuries, head injuries and stroke-related dementia. An exciting new strategy for the treatment of such disorders is to replace the damaged or lost neurons by implanting healthy neurons into the affected areas. For example, the implantation of fetal neurons into the brain of PD patients has resulted in the improvement of motor function. A potential therapeutic approach for treating AD, ALS and spinal cord injuries is to replace lost cholinergic neurons by the implantation of new, healthy cholinergic neurons.
Recent progress in the isolation and propagation of human stem cells in culture has generated a potentially unlimited donor source for such treatments. Current advances in the field include the isolation and propagation of human embryonic stem (ES) cells (Thomson et al. Science 1998; 282:1145) and germ (EG) cells (Shamblott et al. PNAS 1998; 95:13726). These cells are pluripotent, since they can become any cell type in the human body, including neurons. Multipotent neural stem cells have also been isolated successfully from both fetal CNS (see Villa et al. Exp. Neurol. 2000; 161:67-84; Carpenter et al. Exp. Neurol. 1999; 158:265-278; Svendsen et al. J. Neurosci. Methods 1998; 85:141-152); Uchida et al. PNAS 2000; 97:14720-14725; and Vescovi et al. Exp. Neurol. 1999; 56:71-83) or adult CNS (see Roy et al. Nat. Med. 2000; 6:271-277; Johansson et al. Exp. Cell Res. 1999; 253:733-736; and Palmer et al. Nature 2001; 411:42-43). The capabilities of self-renewal and multipotential differentiation render these stem cells an attractive and presumably unlimited donor source for cell replacement therapy to treat neurological disorders.
While such cells could serve as a source of new neurons to ameliorate many neural disorders, a critical issue is how to direct these pluripotent or multipotent stem cells toward a specific cell lineage in order to meet a desirable therapeutic requirement. For example, treating spinal cord injury, AD or ALS using a cell replacement strategy requires the differentiation of cholinergic neurons from these stem cells. Human or rodent stem cells are able to differentiate into specific neuronal types when grafted into either developing CNS (see Flax et al. Nat. Biotechnol. 1998; 16:1033; Brustle et al. Nat. Biotech. 1998; 16:1040-1044; Reubinoff et al. Nat. Biotech. 2001; 19:1134-1140; and Zhang et al. Nat. Biotech. 2001; 19:1129-1133) or neurogenic areas of the adult CNS (see Fricker et al. J. Neurosci. 1999; 19:5990-6005; Shihabuddin et al. J. Neurosci. 2000; 20:8727-8735; and Suhonen et al. Nature 1996; 383:624-627). However, these cells remain undifferentiated or become mainly glial cells when transplanted into non-neurogenic regions of the adult CNS, (see Svendsen et al. Exp. Neurol. 1997; 148:135-146; Sheen et al. Exp. Neurol. 1999; 158:47-62; Fricker et al. J. Neurosci. 1999; 19:5990-6005; Shihabuddin et al. J. Neurosci. 2000; 20:8727-8735; and Cao et al. Exp. Neurol. 2001; 167: 48-58). This indicates that in vitro priming or differentiation prior to grafting is necessary for these cells to become specific neuronal subtypes such as cholinergic neurons. There have been no reports on the generation of a significant number of cholinergic neurons from long-term mitogen-expanded human stem cells. Such neurons play key roles in motor function as well as learning and memory, and thus are highly relevant to clinical applications. It is a major obstacle that the majority of such cells do not differentiate into cholinergic neurons when grafted into non-neurogenic areas of the adult CNS. See Svendsen et al. Exp. Neurol. 1997; 148: 135-146; Sheen et al. Exp. Neurol. 1999; 158:47-62; Fricker et al. J. Neurosci. 1999; 19: 5990-6005; Shihabuddin et al. J. Neurosci. 2000; 20:8727-8735; and Cao et al. Exp. Neurol. 2001; 167: 48-58. Thus, there is a need for new methods to direct pluripotent or multipotent stem cells to differentiate into neurons of a specific lineage. For example, there is a need for methods to produce cholinergic neurons from stem cells and/or progeny thereof.